Publication Date

2014

Document Type

Thesis

Committee Members

Katherine Excoffon (Advisor), Barbara Hull (Committee Member), Dawn Wooley (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Development of latent HIV-infected CD4+ T lymphocytes is the major cause of HIV treatment failure. Adeno-associated virus (AAV) is an attractive vector for anti-HIV gene therapy due to its lack of pathogenicity, low immunogenicity, and persistent transgene expression. However, a major limitation for AAV gene transfer is the cell-specific tropism of each serotype. Only AAV serotypes 2 and 5 have been investigated in hematopoietic cells, both of these serotypes have shown low transduction efficiency. Since each of the currently described AAV serotypes demonstrate distinct tissue tropism, I hypothesized that other serotypes such as AAV1, 4, 8, or 9 may demonstrate improved transduction over AAV2 or 5. To test this, AAV infection of H9 cells (T-cell line), HeLa cells (derived from cervical cancer cells), and primary CD4+ T lymphocytes that were isolated from peripheral blood mononucleated cells (PBMCs) of healthy donors using a ficoll gradient, were investigated. CD4+ T lymphocytes were enriched to ~98% by negative selection using EasysepTM Enrichment Cocktail (Stemcell Tech.). H9 cells, HeLa cells, and CD4+ T lymphocytes were infected at various MOI with various recombinant AAV (rAAV) serotypes encoding the gene for GFP or luciferase. Amongst the different serotypes tested, rAAV2 had the greatest transduction efficiency in H9 cells, rAAV5 had the best transduction efficiency in HeLa cells, while none of the rAAV serotypes appeared to infect CD4+T lymphocytes as determined by fluorescence microscopy, flow cytometry and luciferase assay. Although none of the AAV serotypes investigated demonstrated a transduction efficiency sufficient to achieve a clinically relevant therapeutic index in primary CD4+ T lymphocytes, other serotypes or novel methods to modify tropism might yield vectors suitable for gene delivery in disease-associated leukocytes.

Page Count

96

Department or Program

Microbiology and Immunology


Share

COinS